Ice formation is damaging to living systems and food products and may be a nuisance and a hazard to human beings who must cope with snow and ice in their environment. The field of the present invention is the provision of processes for the preparation of specific chemical agents, referred to herein as ice interface dopants (IID), that will effectively reduce ice formation and make ice that does form innocuous to living systems and foodstuffs and less troublesome and hazardous to humans and machinery in the environment.
Referring to FIGS. 1A-1B, ice crystallizes in the shape of a hexagonal plate 10. A plane defined by the a axis 12 and the b axis 14 (which is crystallographically identical to the a axis) and perpendicular to the c axis 16 defines a hexagonal cross section called the basal plane 18. The six faces of the hexagon are called prism faces 20. Crystallographically, the basal plane 18 is referred to as the 0001 surface, and the prism face is referred to as the 1{overscore (1)}00  surface or the 1{overscore (1)}20  surface depending on the orientation.
FIGS. 2A-2D show that the units of the crystal that give rise to this macroscopic structure are also hexagonal. In FIG. 2A, following common usage, only the oxygen atoms are represented. Stippling indicates oxygens projecting out of the plane of the sheet. The hydrogen atoms lie along the straight lines shown bonding each oxygen atom to its four nearest neighbors.
FIG. 2A shows the basal plane 0001 surface as seen from above. Within each hexagon, three vertices project upward (or forward), and the three intervening vertices project downward (or backward). The upward vertices are separated by 4.5 xc3x85xc2x10.02 xc3x85 and are located at a 60xc2x0 angle with respect to each other. Their fourth bonds extend perpendicularly out of the page toward the viewer. Another spacing at 7.36 xc3x85 separates alternate bilayers 21 of oxygen atoms in the lattice, or, viewed differently, separates each oxygen-defined hexagon from an identical hexagon located immediately adjacent to it. FIG. 2B shows a perspective view of the prism face. Crystallographically, the prism face may be represented as the c1{overscore (1)}00  (FIG. 2C) or d11{overscore (2)}0  (FIG. 2D) prism faces, depending on the angle of the viewer.
Several natural molecules exist that alter the behavior of ice and of water. Antifreeze glycoproteins (AFGPs) and antifreeze proteins or antifreeze peptides (AFPs) produced by several species of fish are believed to adsorb preferentially to the prism face 20 of ice and thus to inhibit ice crystal growth perpendicular to the prism face, i.e, in the direction extending along the basal plane 18 and along the a and b axes 12 and 14.
This capability is sufficient to permit certain fish to live their entire lives at a body temperature about 1xc2x0 C. below the thermodynamic freezing point of the fishes"" body fluids. These fish can ingest and contact ice crystals that might otherwise provide crystal nucleation sites without being invaded by the growth of ice through their supercooled tissues because the AFGPs present in their tissues and body fluids block ice growth despite the presence of supercooling. Insect antifreeze or xe2x80x9cthermal hysteresisxe2x80x9d proteins (THPs) are even more effective, being active at supercooling levels of 2xc2x0 C. or more below the thermodynamic freezing point.
The natural xe2x80x9cantifreezexe2x80x9d or xe2x80x9cthermal hysteresisxe2x80x9d proteins found in polar fish and certain terrestrial insects are believed to adsorb to ice by lattice matching (Davies and Hew, FASEB J., 4; 2460-2468, 1990) or by dipolar interactions along certain axes (Yang, Sax, Chakrabartty and Hew, Nature, 333:232-237, 1988).
AFGPs and AFPs found in certain organisms provide natural xe2x80x9cproofs of principlexe2x80x9d for the concept of novel man-made IIDs. However, natural ice interface doping proteins are not sufficiently active or abundant for most practical applications of interest. Furthermore, a disadvantage of growth inhibition on the prism faces is that, when supercooling becomes sufficient to overcome ice crystal growth inhibition, growth occurs, by default, predominantly in the direction of the c axis 16, perpendicular to the basal plane 18. This results in the formation of spicular or needle-shaped ice crystals (FIG. 1B) that are more damaging to living cells than normal ice, apparently for mechanical reasons. (In FIG. 1B, AFGPs are represented as blobs blocking the prism faces. Other layers are similarly blocked, but the details are omitted for simplicity.)
Natural IIDs are commercially available only in a very limited quantity and variety. Furthermore, they must have fairly high relative molecular masses (typically at least about 4,000 daltons) to be effective. This tends to make them expensive, and they often require complex interactions with other hard-to-acquire proteins and often require carbohydrate moieties for full effectiveness. Insect antifreeze proteins, recently shown to be extremely effective compared to fish antifreeze proteins, still have relative molecular masses of around 8,400 daltons. (Graham, Liou, Walker and Davies, Nature, 388:727-728, 1997).
Furthermore, addition of natural fish AFGP to a concentrated solution of cryoprotectant (30-40% v/v DMSO) had minimal effect on ice crystal growth rates below xe2x88x9220 to xe2x88x9240xc2x0 C. (Fahy, G. M., in Biological Ice Nucleation and its Applications, chapter 18, pp. 315-336, 1995), thus making questionable its effectiveness for use in organ vitrification for cryopreservation.
Another problem with natural antifreeze proteins is that continuing confusion over their precise mechanisms of action hampers the development of recombinant variants that could be more effective. Recently, Warren and colleagues reported some progress in this direction (U.S. Pat. No. 5,118,792).
Caple et al. (Cryo-Letters, 4:51-58, 1983) made several apparently arbitrary synthetic polymers and showed that some of them were able to prevent nucleation of water by silver iodide crystals. They suggested that these polymers adsorbed either to the silver iodide or to ice crystal nuclei, but they did not suggest any specific interactions, and their polymers were made without regard to any consideration of the structure of ice or of AgI. Further, except for noting that a 2 to 1 ratio of hydrophobic to hydrophillic groups on their polymers gave maximum inhibition of nucleation, they provided no guidance or general principles as to how one could approach the synthesis of ice-binding polymers on a systematic theoretical or empirical basis or maximize the ice-binding effectiveness of such polymers. They also taught that higher concentrations of their polymers nucleated their solutions, and failed to teach that their polymers would slow ice crystal growth rates or have other than academic uses. Caple et al. (Cryo-Letters, 4: 59-64, 1983) also reported detecting unidentified, uncharacterized, and unpurified nucleation-inhibiting substances from natural sources, but again suggested no applications.
The concept of designing specific artificial chemical agents whose purpose is to control the physics of ice was first mentioned by Fahy in Low Temperature Biotechnology, McGrath and Diller, eds., ASME, pp.113-146, 1988. The sole mention of this idea was the single statement that xe2x80x9cinsight into the mechanism of AFP action . . . opens the possibility of designing molecules which may be able to inhibit ice crystal growth in complementary ways, e.g., along different crystallographic planes.xe2x80x9d However, no method of preparing such molecules was suggested.
Kuo-Chen Chou (xe2x80x9cEnergy-optimized structure of antifreeze protein and its binding mechanismxe2x80x9d, J. Mol. Biol., 223:509-517, 1992) mentions an intention to specifically design ice crystal growth inhibitors. However, it is confined to minor modifications of existing antifreeze molecules, and does not envision the present radically different approach of preparing synthetic IIDs de novo.
Based on these observations, it is advantageous to design molecules that can inhibit ice crystal growth specifically in the direction of the c axis in accordance with the present invention. When used in combination with an agent acting to block growth in the direction of the basal plane, such that all growth planes would be inhibited rather than only one, such an agent should avoid the lethal drawbacks of the prior art of freezing cells using only basal plane growth inhibitors. Furthermore, since growth in the direction of the c axis, hereinafter xe2x80x9cC growth,xe2x80x9d is the limiting factor for supercooling in the presence of agents that adsorb to the prism face (agents that block growth in the a axis direction, or xe2x80x9cA growthxe2x80x9d), C growth inhibitors should enhance supercooling considerably over the supercooling achievable with A growth inhibitors alone when used in combination with A growth inhibitors.
A problem with natural antifreeze proteins has been continuing confusion over their precise mechanisms of action. Recently, Sicheri and Yang (Nature, 375:427-431, 1995) described a clear model of how AFPs undergo lattice matching with ice. They indicated that, of 8 AFPs examined, the number of ice-binding atoms ranged from 3 to 10 per AFP and that each AFP formed, on average, ice contacts at between 1 in every 4.8 to 1 in every 15 amino acids present in the molecule (roughly 1 ice bond per 422-1340 daltons of AFP mass). The ice-binding amino acids were threonine (thr), aspartate (asp), asparagine (asn), and lysine (lys). Each binding amino acid formed one bond per amino acid and the bonds were formed by the hydroxyl oxygen of thr, the amino nitrogen of lys and of asn, and the acid oxygen (Oxe2x88x92 or carbonyl O) of asp. For the winter flounder AFP, detailed analysis showed that the lattice matching depended on a planar arrangement of the AFP""s bonding groups and on geometrical constraints on the freedom of motion of the matching groups. Bonding took place on the ridges of the 2021 plane (Biophys. J., 63:1659-1662, 1992; Faraday Discuss., 95:299-306, 1993; J. Am. Chem. Soc., 116:417-418, 1994.) More detailed analysis showed that the lattice match between asn and asp oxygen and nitrogen and ice oxygens was imperfect. For one thing, the oxygens in ice associated with these sites were located to the side of each binding atom, not directly underneath. For another, the trigonal planar (sp2) coordination of the hydrogen-bonding groups of asn and asp differ from the tetrahedral (sp3) coordination of oxygens in ice. They concluded that xe2x80x9cthe underlying hydrogen-bonding interactions are likely to be more liberally defined than previously proposedxe2x80x9d by other authors (Biophys. J., 59: 409-418, 1991; Biophys. J., 63:1659-1662, 1992; Biophys. J., 64: 252-259, 1993).
The present invention provides processes for preparing ice interface dopants, ice interface dopants prepared thereby, and methods of using them. One process entails determining a distance between hydrogen bonding sites on an ice nucleating substance and preparing molecules having a complementary bonding distance between their own hydrogen bonding sites and the identified sites on the ice nucleating substance. Enhanced ice bonding capacity of these molecules is obtained by considering in a design process the novel concept of xe2x80x9corbital steering.xe2x80x9d Orbital steering refers to the positioning of lone pair electron orbitals (or hydrogen atoms capable of forming hydrogen bonds) in a preferred direction so as to facilitate hydrogen bonding to ice. This may be accomplished by locking the bonding atoms of the IID into fixed, non-rotating positions by covalently bonding them to at least two other atoms other than hydrogen that form a part of the relatively rigid structure of the IID. Alternately, orbital steering can be accomplished using only rotatable ice-bonding groups whose ice-bonding orbitals are designed to be able to aim directly at hydrogen bonding orbitals of ice. Molecules of the invention may be designed in such a way that they can be both highly active and sufficiently available to be practical to use.
According to the present invention, IIDs can be prepared that exceed the effectiveness of natural agents. Given that nature has been constrained to using protein, which has limited chemical and structural versatility, and limited evolutionary flexibility, non-protein IIDs as provided herein can vastly exceed the performance of existing natural antifreeze macromolecules, provided proper procedures, as provided by the present invention, are followed. By analogy, the fact that insect AFPs are many times more active than fish proteins shows that the principles used by fish proteins can be improved upon. Synthetic IIDs should exceed insect AFP effectiveness by using principles superior to those used by the insect proteins. The present invention provides methods of preparing new and optimal non-protein structures for inhibiting ice crystal formation without regard to existing natural antifreeze proteins or glycoproteins.
The dopant molecules of the present invention can be prepared to adsorb to each surface facet ice presents. Dopant molecules can be prepared to act cooperatively by providing binding sites for other dopant molecules along the edges of the molecule. The invention provides processes for the preparation of molecules that can effectively adsorb to an ice lattice or another ice nucleating surface to preclude ice crystal growth at these ice nucleating surfaces. Additionally, the invention provides processes for the preparation of molecules that can cause nucleation.
The present invention also provides methods for inhibiting the growth of ice in and on various objects, for example, aircraft wings, footwear, pathways, foodstuffs, plants, windows, cables, transplantable organs, tissue, or cells including blood tissues or cells, and other substances or objects where control of ice growth is beneficial.
A process embodied in the invention includes a process for preparing an ice interface dopant comprising determining at least one distance between a plurality of ice crystal template hydrogen bonding sites on a substance capable of nucleating an ice crystal and synthesizing a dopant molecule having a plurality of dopant hydrogen bonding sites spaced at such a distance as to be capable of associating simultaneously with said ice nucleating substance hydrogen bonding sites.
Embodiments also include determining said distance by binding at least one polymer to said ice nucleating substance hydrogen bonding sites, said polymer comprising a polynucleotide that is capable of being amplified directly or indirectly by PCR.
Most particularly, the present invention relates to motifs for ice bonding that allow a) positional matching between hydrogen bonding atoms in ice and of ice-bonding atoms in the IID, b) orbital positioning of those bonding atoms for bonding to hydrogen bonding sites in ice, c) versatility to permit any matching region of ice vertices to be bonded, regardless of whether the locally available ice vertices are projecting hydrogen atoms or oxygen lone pair electron clouds toward the IID, and d) polymerization to form chains that will provide IIDs of desired effectiveness.